The dramatic events of chromosome segregation and cell division are initiated by the activity of cyclin-dependent kinase 1 (Cdk1). The Cdc25 phosphatase promotes entry into mitosis by removing the inhibitory phosphate place on Cdk1 by the Wee1 kinase. Cdc25 and Wee1 are proposed to participate in feedback loops with Cdk1 in order produce an abrupt switch-like activation of Cdk1 and irreversible entry into mitosis. They have also been proposed to participate in signaling networks that integrate information regarding physiological parameters. The mechanisms by which Wee1 and Cdc25 integrate these upstream signals and generate a switch like behavior are poorly understood. The long term goal of the proposed research is to obtain a better understanding of the molecular mechanisms that regulate entry into mitosis via Cdc25. The function of Cdc25 has been shown to be conserved in the budding yeast homolog Mihl Furthermore, we have shown that Mih1 is phosphorylated in vivo and in vitro by Cdk1 and casein kinase 1;however, it us unclear how phosphorylation by these two kinases regulate Mini. To further our studies, we will characterize the mechanisms by which Cdk1 and casein kinase 1 phosphorylation regulate Mihl activity by addressing the following specific aims: (1) phosphorylation site mapping and mutagenesis will be used to test the hypothesis that Cdk1 activates Mihl in a positive feedback loop that promotes entry into mitosis;(2) a combination of genetics and biochemistry will be used to determine how casein kinase 1 regulates Mini;(3) fluorescence microscopy will be used to test the hypothesis that nuclear localization plays a role in regulating the phosphorylation state of Mihl. The mechanisms that regulate Mih1/Cdc25 are of considerable interest because they represent potential targets for drugs aimed at blocking the division of cancer cells. In addition, the mechanisms that generate switch like responses are of fundamental importance to our understanding of the diverse signaling networks that are the target of many known drugs.